Anat Cell Biol 2020; 53(4): 471-480
Published online December 31, 2020
Copyright © Korean Association of ANATOMISTS.
1Department of Internal Medicine, Dongguk University Gyeongju Hospital, Gyeongju, 2Division of Pulmonary, Allergy and Critical Care Medicine, Department of Internal Medicine, Inje University Busan Paik Hospital, Busan, 3Department of Anatomy and Tumor Immunology, Inje University College of Medicine, Busan, Korea
Correspondence to:Dae Young Hur
Department of Anatomy and Tumor Immunology, Inje University College of Medicine, Busan 47392, Korea
*These two authors contributed equally to this work.
Over-expression of nicotinamide adenine dinucleotide phosphate oxidase (Nox) isoform enzymes was recently reported in various cancers including Burkitt’s lymphoma (BL). However, the functions of Nox isoform enzymes in BL remain poorly understood. In this study, Nox isoform expression and the effects of a Nox-specific inhibitor were evaluated in Epstein-Barr virus (EBV)-positive Raji BL cells in comparison with EBV-negative Ramos BL cells. To evaluate Nox enzyme expression in Raji and Ramos BL cells, polymerase chain reaction (PCR) and western blot analysis were performed. To verify the intracellular signaling mechanism of the Nox inhibitor-induced apoptosis of Raji cells, WST-1 assay, trypan blue exclusion method, flow cytometry, PCR, western blotting, and bromodeoxyuridine staining were conducted. Experiments using the pan-caspase inhibitor z-VAD, reactive oxygen species scavenger N-acetyl-L-cysteine (NAC), and Bim inhibitor 1 were performed. PCR and western blot results showed that Nox isoform enzymes were highly expressed in EBV-positive BL Raji cells compared with EBV-negative BL Ramos cells. The Nox2 inhibitor induced apoptosis of Raji cells in time- and dose-dependent manners. The Nox2 inhibitor also caused up-regulation of Bim and Noxa, down-regulation of Mcl-1, translocation of Bax, release of cytochrome
Keywords: Nicotinamide adenine dinucleotide phosphate oxidase, Burkitt lymphoma, Epstein-Barr virus, Apoptosis
Burkitt’s lymphoma (BL) is a highly aggressive and invasive B-cell non-Hodgkin lymphoma with varying patterns of clinical behavior . BL patients are slowly decreasing and cure rates also improved. However, elder patients still have poor prognosis . BL is associated with Epstein-Barr virus (EBV) infection and shows clinical symptoms that have been categorized into sporadic, endemic, and immunodeficiency-associated subtypes. BL is characterized by translocations of chromosomes 8 and 14 that result in upregulation of the c-myc protein and subsequent increase of cell proliferation [3, 4]. With recent improvements in chemotherapy regimens and the application of new targeted therapies, the chance for successful treatment of children with BL has significantly improved [5, 6]. However, elderly patients and patients with advanced disease often show invasion to the central nervous system, and the prognosis for these patients is poor [7-9]. Therefore, the identification of an effective new treatment method for BL is urgently required. The development of a molecular-targeted therapy has presently taken center stage as the preferred treatment option.
Raji cells are derived from the B-lymphocytes of a male BL patient and categorized as lymphoblast-like. Some characteristics of Raji cells include a lack of differentiation, illustrated by the formation of large aggregations of hundreds of individual cells. Raji cells produce an unusual strain of EBV, which both transforms cord blood lymphocytes and induces early antigens in the cells [10, 11]. Ramos cells originated from a human Caucasian BL male patient, and are EBV-genome-negative . In the present study, these two cell lines were used as representative cell lines for BL.
EBV is a herpes virus type 4 which contains about 172 kilobase double strand DNA genome encoding about 100 products and non-coding RNAs. Latent viral proteins such as latent membrane proteins (LMPs) are associated with latent program in a host and deeply related to intracellular signal transduction, cellular regulation and cell proliferation of host cells as well as maintaining EBV genome .
Reactive oxygen species (ROS) plays an important role in various cell functions including the process of virus-induced malignant transformation [14, 15]. A recent study reported that the increase in ROS level by EBV infection provides a growth advantage to EBV-infected cells by inducing the expression of cell proliferation-associated genes . Regarding the role of ROS in B cell lymphoma including BL, another study reported that proliferation was induced through ROS increase . Therefore, ROS is a potential therapeutic target in virus-associated cancers including BL. LMP1 regulates the expression Nox and Nox regulatory subunit p22phox .
The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) family is composed of the Nox1, -2, -3, -4, -5 and dual oxidase 1, -2 proteins and includes the major enzymes responsible for the production of ROS [18, 19]. Over-expression of Nox isoform enzymes was recently reported in colorectal cancer and non-small cell lung cancer [20, 21]. Furthermore, these enzymes play an important role in cell cycle regulation and have been proposed as a potential target in BL [22, 23]. However, the effects of the specific inhibitors for Nox isoforms and the signaling mechanisms remain poorly understood.
The Bcl-2 family proteins are major regulators of apoptosis [24, 25]. The pro-survival members (Bcl-2, Bcl-xL, Mcl-1, and A1) are integral for cell survival; the BH3-only proteins (Bim, Puma, Bad, Noxa) initiate apoptosis signaling; and Bax/Bak are required for mitochondrial outer membrane permeabilization [25-27]. The conformational changes of Bax and Bak lead to homo-oligomerization of the proteins and the formation of outer-mitochondrial membrane spanning pores . These Bax and Bak pores permit the release of cytochrome
In this study, we evaluated the expressions of Nox isoforms and the effects and cell signaling mechanism of a specific Nox inhibitor in EBV-positive Raji BL cells in comparison with EBV-negative Ramos BL cells.
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll-paque gradient centrifugation (Amersham Life Science, Buckinghamshire, UK). Primary B cells were purified from PBMCs using a magnetic-activated cell sorting B cell-negative depletion kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Ramos, an EBV-negative B lymphoma cell line, and Raji, an EBV-positive B lymphoma cell line, were obtained from the American Type Culture Collection (Rockville, MD, USA). Both cell lines were maintained in RPMI-1640 medium (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (HyClone) and antibiotics at 37°C in humiditied 5% CO2 atmosphere.
Western blot was performed using the following primary antibodies: anti-human Nox2, anti-human Nox4 (BD Biosciences, San Diego, CA, USA), and anti-human β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). GSK2795039, a Nox2 inhibitor, was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Anti-human caspase 3 and 9 antibodies (Abcam, Cambridge, MA, USA) and anti-human Mcl-1, Bcl-2, Bim, Noxa antibodies (Cell Signaling Technology, Beverly, MA, USA) were used to evaluate protein expression. N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone (Z-VAD-FMK, Sigma, St. Louis, USA), a pan-caspase inhibitor; N-acetyl-L-cysteine (NAC, Cell Signaling Technology), a ROS scavenger; and ABT-263 (Selleck Chemical, Shanghai, China), a Bim specific inhibitor were used to identify the intra-cellular signaling pathway.
Cells were collected and washed three times with phosphate-buffered saline (PBS, pH=7.4). Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was transcribed into cDNA using the reverse-transcription PreMix Kit (Bioneer, Daejeon, Korea). Polymerase chain reaction (PCR) amplification was performed using primer sets (Bioneer) specific for Nox-2 (Forward: 5’-CAT GTT TCT GTA TCT CTG TGA-3’; Reverse: 5’-GTG AGG TAG ATG TTG TAG CT-3’) and Nox-4 (Forward: 5’-CCA TGG CTG TGT CCT GGA GGA GCT G-3’; Reverse: 5’-AGT TGA GGG CAT TCA CCA GAT GGG C-3’). For the control, a specific primer set for β-actin (Forward: 5’-ATC CAC GAA ACT ACC TTC AA-3’; Reverse: 5’-ATC CAC ACG GAG TAC TTG C-3’) was used, which yielded a 200-bp product. PCR products were visualized on 2.5% agarose gels with ethidium bromide. The normalization and fold-changes of levels of Nox2 and Nox4 expression were calculated with Image J 1.38 software (http://rsweb.nih.gov/ij/index.html). Each experiment was repeated at least three times.
Cells were harvested and washed twice with PBS. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Elpis Biotech, Daejeon, Korea) containing a protease inhibitor cocktail (AEBSF, aprotinin, bestatin hydrochloride, E-64, EDTA, and leupeptin hemisulfate salt; Sigma). To evaluate phosphorylation events, an additional set of phosphatase inhibitors were added to the RIPA buffer (Cocktail II; sodium orthovanadate, sodium molybdate, sodium tartrate, and imidazole; Sigma). Protein concentration was estimated with the bicinochoninic acid Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of proteins (40 μg) were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham) by immunoblotting. The membranes were then incubated overnight at 4°C in a PBS solution supplemented with 5% non-fat dry milk. The blots were probed with specific primary antibodies for 1 hour, incubated with diluted enzyme-linked secondary antibody, and then visualized by enhanced chemiluminescence as recommended by the manufacturer (Amersham). Equivalent protein loading was assessed by β-actin expression. Each experiment was repeated at least three times.
Cells were seeded and then treated with GSK2795039 at the indicated doses for 48 hours, and cell viability was analyzed using a WST-1 assay (Sigma) or trypan blue exclusion method.
Cells were harvested, washed twice with PBS and resuspended in 100 ml Annexin V binding buffer (10 mM HEPES, 0.14 M NaCl, and 0.25 mM CaCl2). Next, 2 ml FITC-conjugated Annexin V (BD Biosciences) and 1 ml PI (BD Biosciences) were added, and cells were incubated at room temperature for 15 minutes in the dark with gentle vortexing. Next, 400 ml of Annexin V binding buffer was added to each tube, and cells were analyzed using a flow cytometry.
Mitochondrial and cytosolic fractions of cells were prepared using a Cytosol/Mitochondria fractionation kit (Calbiochem, San Diego, CA, USA). Briefly, cells were harvested by centrifugation at 600×g for 5 minutes at 4°C and washed twice with cold PBS. Cells were then resuspended in 250 ml cytosol extraction buffer containing protease inhibitor mixture (included in the fractionation kit) and 1 mM dithiothreitol (DTT). After incubation on ice for 10 minutes, cells were homogenized using a Dounce tissue homogenizer on ice. Homogenates were centrifuged at 700×g for 10 minutes at 4°C, and the supernatant was centrifuged again at 10,000×g for 30 minutes at 4°C. The resulting supernatants were harvested and designated as cytosolic fractions. The pellets were resuspended in 50 ml mitochondria extraction buffer containing protease inhibitor mixture and DTT and designated as mitochondrial fractions. The fractionated cell lysates (10 mg per well) were prepared for western blotting using anti-Cox IV and anti-beta tubulin for loading control.
To detect the sub-G1 peak, PI staining was performed (BD Biosciences). Briefly, cells were incubated with Nox2 inhibitor, z-VAD, NAC, or Bim inhibitor in culture medium. Cells were collected and washed according to the instructions supplied by the manufacturer. Cell pellets were stained with PI staining solution containing RNase A (10 mg/ml; Sigma) and PI (2 mg/ml) in PBS. The cell suspension was then incubated in the dark at room temperature for 20 minutes. DNA content was determined using a FACSCalibur flow cytometer (BD Biosciences).
All data are expressed as mean±standard deviation and each value represents at least two different experiments. Comparisons between all individual data were made using one-way ANOVA. Statistical significance was defined by a
Raji is an EBV-positive B lymphoma cell line, and Ramos is an Epstein-Barr-negative B lymphoma cell line. Recent studies reported that these two cell lines showed different characteristics including phenotypes [9-11]. Nox2 and Nox4 expression were first evaluated in primary B, Raji, and Ramous cells. PCR (Fig. 1A) and western blotting (Fig. 1B) results showed that Nox2 mRNA and protein were highly expressed in Raji cells compared with the other cell lines. However, Nox4 protein only was highly expressed in Raji cells compared with the other cells.
Our results showed that Raji cells significantly expressed Nox2 mRNA and protein as evaluated by PCR and western blotting. To assess the role of Nox2, the cellular responses of Raji cells were observed after treatment using a specific inhibitor for Nox2, GSK2795039. The Nox 2 inhibitor significantly induced cell death in only Raji cells in a dose-dependent manner. The most effective concentration of GSK2795039 was 12.5 μM concentration, and Raji cells showed the similar effects in higher concentration (Fig. 2B). GSK2795039 had no effect on primary B cells (Fig. 2A) and induced slight cell death in Ramos cells only at a high concentration; however, the induction of cell death was not statistically significant (Fig. 2C).
Our results showed that Nox2 inhibitor effectively induced cell death in only Raji cells. We next examined whether Nox2-induced cell death was associated with apoptosis. Cells treated with Nox2 inhibitor were stained with FITC-labeled Annexin V and PI and analyzed by flow cytometry. Nox2 inhibitor treatment induced the apoptosis of Raji cells in a time-dependent manner (Fig. 3). Nox2 inhibitor treatment also induced apoptotic cell death in Raji cells in a dose-dependent manner, but no effects were observed in Ramos cells. The difference in Nox2 inhibitor induced-apoptosis between Raji and Ramos cells were statistically significant at 6.25 and 12.5 μM concentrations (Fig. 4A).
Our results showed that the Nox2 inhibitor induced apoptosis in Raji cells. Caspases are key effector molecules that induce apoptosis in cancer cells . Therefore, we next evaluated the activation of caspases after Nox2 inhibitor treatment in Raji cells. The results showed that the Nox2 inhibitor induced the cleavage of caspases 3 and 9 (activated forms) in Raji cells in a dose-dependent manner, but no effects were observed in Ramos cells (Fig. 4B).
To identify the mechanism of Nox2 inhibitor-induced apoptosis, several candidate signaling molecules were evaluated by western blot. Mcl-1, Bcl-2, Bim, and Noxa were expressed constitutively in both Raji and Ramos cells; GSK2795039 treatment decreased Mcl-1 expression and increased Bim and Noxa expression in Raji cells in a dose-dependent manner, however, GSK2795039 had no effect on Ramos cells, similar to the previous results (Fig. 5).
Next, we examined translocation of Bax, a pro-apoptotic protein, from the cytoplasm to mitochondria and release of Cyt
To clarify the signaling pathway of Nox2 inhibitor induced-apoptosis in Raji cells, inhibition assays were performed using the pan-caspase inhibitor z-VAD, the ROS scavenger NAC, and the Bim inhibitor 1 BI-1. z-VAD, NAC, and BI-1 effectively blocked the translocation of Bax and release of Cyt
Several previous studies have demonstrated that tumor virus infection is associated with increased oxidative stress through Nox induction, which exerts an important role in virus-induced transformation [29-31]. A recent study reported that EBV increases the proliferation of EBV-infected cells through the induction of ROS by cellular growth-associated factors in B lymphocytes .
In this study, the relationship between ROS-generating enzymes and EBV infection was assessed in two BL cell lines. We first investigated whether EBV infection could regulate the expression of the ROS-generating Nox enzyme family using EBV-positive Raji and EBV-negative Ramos BL cells. Our results showed that both Nox2 and Nox4 were highly expressed in EBV-positive Raji cells compared with EBV-negative Ramos cells. However, Nox2 was more highly expressed than Nox4 in Raji cells as shown by PCR and western blotting. Moreover, the Nox2 specific inhibitor GSK2795039 induced cell death only in Raji cells in a dose-dependent manner. The most effective concentration of GSK2795039 was 12.5 μM concentration, therefore this concentration was used in the further experiments. These results suggested that Nox2 expression depends on EBV infection and could be a possible target for treatment in EBV-positive BL tumors. A recent study reported that the EBV oncoprotein LMP1 regulates the expression of Nox and a Nox regulatory subunit . This report also supports the relationship between Nox enzymes and EBV infection.
We next evaluated whether apoptosis was involved in the cell death of Raji cells by GSK2795039 treatment using an Annexin-V/PI assay kit. GSK2795039 treatment induced the apoptosis of Raji cells in time- and dose-dependent manners, but no effect was observed in Ramos cells. In addition, GSK2795039 treatment induced the activation of caspase 3 and 9 in Raji cells in a dose-dependent manner. The pan-caspase inhibitor z-VAD completely inhibited Nox inhibitor-induced apoptosis of Raji cells. These results indicated that GSK2795039 treatment induced the apoptosis of Raji cells in a caspase-dependent pathway.
Caspase activation is regulated at the multi-phase, including the release of other proteolytic enzymes such as Cyt
In this study, Nox2 inhibitor, GSK2795039, induced down-regulation of Mcl-1 and up-regulation of Bim and Noxa in a dose-dependent manner (as shown in Fig. 5). GSK2795039 induced the translocation of Bax and release of Cyt
In summary, Nox enzymes were highly expressed in EBV-positive Raji cells. The Nox specific inhibitor induced up-regulation of Bim and Noxa, down-regulation of Mcl-1, translocation of Bax, release of Cyt
This work was supported by the 2019 Inje University research grant.
Conceptualization: DYH. Data acquisition: CHR, SHK. Data analysis or interpretation: CHR, DYH. Drafting of the manuscript: CHR. Critical revision of the manuscript: DYH. Approval of the final version of the manuscript: all authors.
No potential conflict of interest relevant to this article was reported.